Project Summary/Abstract Thymidine kinase 2 (TK2), encoded by the nuclear gene, TK2, is a mitochondrial matrix protein that phosphorylates thymidine and deoxycytidine nucleosides to generate deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP), which in turn, are converted to deoxynucleotide triphosphates (dNTPs) required for mitochondrial DNA synthesis. Autosomal recessive TK2 mutations cause devastating neuromuscular weakness with severe depletion of mitochondrial DNA (mtDNA) in infants and children, as well as progressive external ophthalmoplegia with mtDNA multiple deletions in adults. The central nervous system is variably involved in these disorders. To determine the bases of disease onset and organ-specificity of TK2 deficiency, Michio Hirano, Drs. Orhan H Akman, and collaborators have generated a homozygous Tk2 H126N knock-in mutant (Tk2-/-) mouse that manifests a phenotype strikingly similar to the human infantile encephalomyopathy. Between postnatal day 10 and 13, Tk2-/- mice rapidly develop fatal encephalomyopathy characterized by decreased ambulation, unstable gait, coarse tremor, growth retardation, and rapid progression to early death at age 14 to 16 days. Molecular and biochemical analyses of the mouse model demonstrated that the pathogenesis of the disease is due to loss of enzyme activity and ensuing dNTP pool imbalances with decreased dTTP levels in brain and both dTTP and dCTP levels in liver, which, in turn, produces mtDNA depletion and defects of respiratory chain enzymes containing mtDNA- encoded subunits, most prominently in the brain and spinal cord. We have demonstrated that molecular bypass therapy with orally administered dCMP and dTMP ameliorates the abnormal phenotype and extends the lifespan of Tk2-/- mice by 2-3 fold, but does not cure the disease. Therefore, more effective therapies are needed to treat TK2 deficiency. Based on our unexpected observation that dTMP and dCMP supplementation increased blood and tissue levels of deoxythymidine (dT), we hypothesize that increasing dT and deoxycytidine (dC) substrates for TK2 will be therapeutic. An alternative treatment approach is gene therapy. Taking advantage of our Tk2 knockin mouse model, this proposal seeks to test both therapeutic approaches: 1) pharmacological treatment with deoxynucleosides and 2) gene therapy using adeno-associated virus (AAV) vector-mediated TK2 delivery.